Saliva is critical for the maintenance of oral function and health. Radiation treatment of head and neck cancer patients causes cellular damage in the salivary glands that is usually severe, irreversible, and renders them atrophic. Permanent repair of such damage is the next medical challenge in therapy, and could potentially be accomplished by cell-replacement. The long range goal of this proposal is to identify the source of progenitor cells in the salivary gland and to use these cells for the restoration of salivary function in an impaired gland. We propose that cells from the ductal fraction of the salivary parenchyma should include the uncommitted progenitor cells, and would therefore be a well suited donor cell population to regenerate an atrophic salivary gland after cell transplantation. To facilitate the identification of such a cell population, we have generated a novel transgenic mouse line, which expresses a fusion protein of EGFP and Cre recombinase specifically in the duct cells of the salivary glands. EGFP expression acts as a cell-specific marker of duct cells in these mice. In addition, ductal cell fates can be monitored by staining for LacZ after crossing with mice from the ROSA26 reporter line. Activation of the LacZ reporter gene by recombinase is maintained in a cell's progeny, tracing the cell lineage. Our transgenic strain represents a unique resource on which our experimental plan is based. We propose two specific aims: (1) To identify a subpopulation of salivary gland cells which includes progenitor cells capable of reconstituting multiple salivary cell types. Cells from proliferating submandibular glands will be separated into two populations using fluorescence activated cell sorting: EGFP-positive duct cells, and EGFP-negative acinar cells. The cell types which arise from each pool in vitro will be analyzed to determine whether progenitor cells do reside in the ductal fraction. These experiments will test the hypothesis that duct cells include multipotent precursor cells that are suitable for repopulation of atrophic salivary glands and will establish optimal conditions for their cultivation. (2) Introduce cultured precursor cells into a damaged salivary gland, and establish an assay for the correct functioning of these cells. Proliferating cells will be introduced into atrophic mouse salivary glands. Cell survival and proliferation will be monitored using immunohistochemistry. To evaluate the ability of the transplanted cells to functionally contribute to the recipient organ, we will generate a transgenic line expressing a tagged human salivary protein, the expression and secretion of which can be readily monitored. Cells isolated from this line will be injected and secretion will be measured. The identification of a defined progenitor cell population and demonstration of its ability to repopulate and function in the salivary gland is a critical step in determining the efficacy of cell transplantation as a potential treatment for the atrophic organ. [unreadable] [unreadable] [unreadable]